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This study investigates the uptake and retention of stable fluorescent organosilica nanoparticles by macrophages, which play a vital role in scavenging environmental nanoparticles and nanomedicines within the body. We used rhodamine 6G-loaded fluorescent organosilica nanoparticles (SiNP-R6G) synthesized from a thiol-functionalized organosilane precursor. Our primary objective was to establish a quantitative relationship between fluorescent measurements and nanoparticle tracking analysis, enabling the precise "counting" of nanoparticles taken up by macrophages under kinetic measurement conditions. Our kinetic study demonstrated a concentration-dependent, saturable internalization of nanoparticles in a model macrophage (RAW 264.7 cells), with a maximum uptake rate (Vmax) of 7.9 × 104 nanoparticles per hour per cell. The estimated number concentration of nanoparticles for half-maximum uptake was approximately 0.8 trillion nanoparticles per milliliter, and a significant portion (∼80%) of internalized SiNP-R6G remained entrapped within the cells for 48 h, indicating the sustained particle retention capacity of macrophages. Our data further demonstrate that the internalized nanoparticles occupy more than 10% of the total cell volume, without inducing cytotoxic effects. This heightened capacity for nanoparticle uptake is attributed to intracellular vesicles capable of accommodating up to 50 densely packed, yet nonaggregated, nanoparticles within a single vesicle. In summary, our study underscores the effective development of a methodology for precise "counting" of cellular nanoparticle uptake in macrophages, providing valuable insights into the kinetics and retention capabilities of macrophages for nanoparticle-based drug delivery. [ABSTRACT FROM AUTHOR]

Secondary metabolites in marine organisms exhibit various pharmacological activities against diseases, such as cancer. In this study, the anti‐proliferative effect of JBIR‐100, a macrolide isolated from Streptomyces sp., was investigated in breast cancer cells. Cell growth was inhibited in response to JBIR‐100 treatment concentration‐ and time‐dependently in both MCF‐7 and MDA‐MB‐231 breast cancer cells. JBIR‐100 caused apoptosis, as verified by caspase activation and the cleavage of PARP. Western blotting revealed that JBIR‐100 modulated the expression of Akt/NF‐κB signaling components and Bcl‐2 family members. Overexpression of Mcl‐1 partially rescued MCF‐7 cells from JBIR‐100‐induced cytotoxicity. In addition, transmission electron microscopy analyses, confocal analysis, and western blot assay indicated that JBIR‐100 inhibited autophagy in MCF‐7 cells. Exposure to the autophagy inhibitor did not synergize JBIR‐100‐induced apoptosis. In summary, our results suggested that JBIR‐100 may be potentially used for breast cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract: 5′ AMP‐activated protein kinase enzyme (AMPK), a master regulator of cellular metabolism, is recognized for its association with various metabolic diseases, inflammation and cancer. In this study, we aimed to investigate the role of compound 59, an AMPK activator, in a panel of oral squamous cell carcinoma (OSCC) cell lines. The antiproliferative effects of compound 59 were assessed by MTT assays, flow cytometry, Western blotting, confocal microscopy and transmission electron microscopy. Relative to OSCC cells, normal human oral keratinocytes were almost insensitive to compound 59 treatment. Compound 59 induced apoptosis as indicated by caspase activation and PARP cleavage. In addition, it inhibited JAK/STAT3 signalling, arrested cells in the G1 phase, increased reactive oxygen species (ROS) generation and promoted autophagy. Interestingly, pre‐treatment with a protein tyrosine phosphatase (PP2A) inhibitor, cantharidin, partially reversed compound 59‐induced down‐regulation of p‐JAK2 and p‐STAT3, thereby suggesting the involvement of a protein tyrosine phosphatase. Together, these findings substantiate the potential of compound 59 for the treatment of OSCC patients. [ABSTRACT FROM AUTHOR]

A mixed organosilane system for simultaneous formation of organosilica nanoparticles has been systematically studied for loading of various compounds with a wide range of log P values. The molecule-entrapping system was understood by investigating the effects of adjusting various experimental parameters on particle formation and molecule entrapment. Particularly, rhodamine 6 G (R6G) loaded colloidal particles were prepared and characterised in detail. The results show that whereas most molecules had entrapment efficiency (EE%) in the range of 20-80%, R6G exhibited near 100% efficiency. Moreover, the colloidal system can be tuned to incorporate R6G with the extent of entrapment spanning at least 2 orders of magnitude (i.e. from 0.04 to 4 mg) and a maximum EE% of 98%. In conclusion, the study demonstrates the promise of the proposed mixed organosilane system in forming colloidal particles containing multiple functional groups with selective loading of highly hydrophobic molecules. [ABSTRACT FROM AUTHOR]

Dye-loaded, fluorescent nanoparticles (FNPs) have been extensively studied as promising imaging or probing agents for therapeutic and biomedical applications, because of improved photostability and reduced cytotoxicity (compared with free dyes). The synthesis of FNPs often involves entrapment of fluorescent dye molecules into nanostructures, which, however, would encounter a problem associated with the formation of molecular aggregates within the confined matrix space. The formation of nonfluorescent aggregates seems unavoidable for some conventional fluorescent dyes, thereby leading to fluorescent quenching. The problem is well-recognized, but frequently ignored; and FNPs are usually applied as prepared without addressing it either during or after the synthesis of FNPs. The ignorance may be due to the difficulty in altering post-synthetically the intraparticulate molecular arrangements and interactions. Herein, we describe how light-responsive S-nitrosothiol (SNO) can be engineered into fluorophore-loaded silica nanoparticles for efficient dye-loading and post-synthesis fluorescence augmentation. Silica nanoparticles loaded with various fluorescent molecules were prepared using one-pot, simultaneous, acid-catalyzed sol–gel condensation and nitrosation of a single mercaptosilane source, 3-mercaptopropyl trimethoxysilane (MPTMS), followed by nanoprecipitation. We first show how doxorubicin-loaded silica NPs respond to stepwise visible-light exposure and exhibit ON/OFF fluorescent response. We then demonstrate that rhodamine 6G (R6G) can be stably incorporated into SNO-enriched silica nanostructure with negligible payload leakage (＜0.2%) and ∼1000-fold reduction in cytotoxicity (compared with free R6G). Remarkably, visible light irradiation leads to ∼100-fold increase in fluorescent intensity. Thus, for the first time, SNO is proposed as a light-responsive entity for post-synthesis fluorescence intensification in FNPs. [ABSTRACT FROM AUTHOR]

Codelivery of nitric oxide (NO) and drugs based on a single nanocarrier is a promising therapeutic strategy. Here, we report a one-step nanoprecipitation method to generate nanoparticles that possess simultaneous NO-donating and doxorubicin releasing properties. S-Nitroso polysilsesquioxane acts like an avid “drug sponge” that attracts drug molecules into nanospheres. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is an important molecule that exerts multiple functions in biological systems. Because of the short-lived nature of NO, S-nitrosothiols (RSNOs) are believed to act as stable NO carriers. Recently, sulfhydryl (SH) containing macromolecules have been shown to be promising NO carriers. In the present study, we aimed to synthesize and characterize a potential NO carrier based on bovine Cu,Zn-superoxide dismutase (bSOD). To prepare S-nitrosated bSOD, the protein was incubated with S-nitrosoglutathione (GSNO) under varied experimental conditions. The results show that significant S-nitrosation of bSOD occurred only at high temperature (50 °C) for prolonged incubation time (>2 h). S-nitrosation efficiency increased with reaction time and reached a plateau at ~4 h. The maximum amount of NO loaded was determined to be about 0.6 mol SNO/mol protein (~30% loading efficiency). The enzymatic activity of bSOD, however, decreased with reaction time. Our data further indicate that NO functionality can only be measured in the presence of extremely high concentrations of Hg2+ or when the protein was denatured by guanidine. Moreover, mildly acidic pH was shown to favor S-nitrosation of bSOD. A model based on unfolding and refolding of bSOD during preparation was proposed to possibly explain our observation. [ABSTRACT FROM AUTHOR]

To study oxidative stress in biological systems, chemical compounds capable of producing free radicals have been widely used. Here, we compared two free-radical generators, 3-morpholinosydnonimine (SIN-1) and 2,2′-azo-bis(2-amidinopropane) hydrochloride (AAPH), by measuring the thiol oxidation kinetics of various thiols. We found that SIN-1 is > 30 times potent in causing thiol oxidation than AAPH. Kinetic simulations revealed that in the SIN-1 system (0.1 mM), superoxide, nitrogen dioxide and carbonate radicals are the major reactive species which, in combination, induce ∼50% of thiol molecules to undergo one-electron oxidation, thereby forming the thiyl radical which propagates further thiol oxidation by direct coupling with thiolates. Similarly, the alkyl peroxyl radical derived from AAPH (3 mM) initiates comparable extent of one-electron oxidation and formation of the thiyl radical. In conclusion, our study provides experimental and theoretical evidence that SIN-1 is mainly an one-electron oxidizing agent that can be functionally mimicked by AAPH. [ABSTRACT FROM AUTHOR]

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn't inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs. [ABSTRACT FROM AUTHOR]